PROCEEDINGS OF THE LATVIAN ACADEMY OF SCIENCES. Section B, Vol. 67 (2013), No. 2 (683), pp. 116–123. DOI: 10.2478/prolas-2013-0018

OCCURRENCE OF STONE FRUIT IN PLUM ORCHARDS IN LATVIA Alina Gospodaryk*,**, Inga Moroèko-Bièevska*, Neda Pûpola*, and Anna Kâle*

* Latvia State Institute of Fruit-Growing, Graudu iela 1, Dobele LV-3701, LATVIA [email protected] ** Educational and Scientific Centre „Institute of Biology”, Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., Kiev 01033, UKRAINE

Communicated by Edîte Kaufmane

To evaluate the occurrence of nine viruses infecting Prunus a large-scale survey and sampling in Latvian plum orchards was carried out. Occurrence of mosaic (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), and Plum pox virus (PPV) was investigated by RT-PCR and DAS ELISA detection methods. The de- tection rates of both methods were compared. Screening of occurrence of latent ringspot virus (SLRSV), Arabis (ArMV), Tomato ringspot virus (ToRSV) and Petunia asteroid mosaic virus (PeAMV) was performed by DAS-ELISA. In total, 38% of the tested trees by RT-PCR were infected at least with one of the analysed viruses. Among those 30.7% were in- fected with PNRSV and 16.4% with PDV, while ApMV, ACLSV and PPV were detected in few samples. The most widespread mixed infection was the combination of PDV+PNRSV. Observed symptoms characteristic for PPV were confirmed with RT-PCR and D strain was detected. Com- parative analyses showed that detection rates by RT-PCR and DAS ELISA in plums depended on the particular virus tested. The results obtained in this study revealed that commonly grown plum cultivars in Latvia are infected with economically important stone fruit viruses and highlight the need to implement a programme to produce and propagate virus-free planting material. Key words: Prunus viruses, DAS-ELISA, RT-PCR, detection rates.

INTRODUCTION (García and Cambra, 2007). PPV is considered as the most damaging virus affecting stone fruits and causing yield Stone fruits are hosts for a large number of viruses that can losses up to 80–100% (Nemeth, 1986; Cambra et al., 2006). cause substantial economic losses (Nemeth, 1986; Desvig- PPV is transmitted by vegetative propagation and natural nes, 1999; Myrta et al., 2003). Among the viruses affecting vector aphid species. The use of infected plant material for plums, Prunus necrotic ringspot virus (PNRSV), Prune propagation is the main cause of long-distance spread, dwarf virus (PDV), and Plum Pox virus (PPV) are the most whereas at a site, spread is mediated by a number of aphid significant with worldwide distribution. PNRSV, PDV and species in a non-persistent manner (Labonne et al., 1995; Apple mosaic virus (ApMV) are members of the genus Isac et al., 1998). PPV isolates are currently grouped into , family (Roosinck et al., 2005). seven distinct strains, which differ in their pathogenicity, These viruses, alone or in combination, can severely affect host range, serological and molecular characteristics, and fruit yield, fruit maturity, and tree growth (Uyemoto et al., geographical distribution (Glasa and Candresse, 2005; 1992). Apple chlorotic leaf spot virus (ACLSV) (Tricho- García and Cambra, 2007; Barba et al., 2011). Most PPV virus, ) is economically important due to its isolates belong to the D, M and Rec types. PPV-M is mostly high prevalence and to the detrimental effects caused in known in Southern, Eastern and Central Europe, while some infected stone fruit species and rootstocks. Infection PPV-D mostly in Western Europe (Sochor et al., 2012). of these viruses is often symptomless in most of the stone fruit cultivars. ACLSV, together with PNRSV, may give rise to chlorotic “oak leaf” like patterns on plum leaves Other less prevalent viruses that infect Prunus species be- (Myrta et al., 2011). long mainly to the families and , such as Strawberry latent ringspot virus (SLRSV, Sadwa- PPV (, ) is the causal agent of Sharka virus), (ArMV, ) and Tomato disease on Prunus spp. worldwide, including economically ringspot virus (ToRSV, Nepovirus). Petunia asteroid mo- important stone fruit crops, such as , apricot and plum saic virus (PeAMV, ) belongs to the family

116 Proc. Latvian Acad. Sci., Section B, Vol. 67 (2013), No. 2. Tombusviridae. These viruses are listed in the European and from 28 commercial plum orchards and germplasm collec- Mediterranean Plant Protection Organisation (EPPO) stan- tions in five regions of Latvia. The samples were collected dards as a requirement to be tested in the certification from randomly selected trees without symptoms, except in a scheme for cherry varieties and rootstocks (Anonymous, few cases when typical viral infection symptoms were pres- 2004). In Prunus, ToRSV can cause serious diseases includ- ent. Samples were placed in plastic bags in an ice box and ing stem pitting and decline in peach and cherry, yellow bud transported to the laboratory where they were immediately mosaic in peach and almond and brown line and decline in analysed or frozen in liquid nitrogen and stored at –80 °C plum. PeAMV infection may cause 5–30% loss of the mar- until use. Before analyses or preservation each leaf in the ketable value of the fruits due to malformations with ne- sample was divided in two portions to equally represent the crotic spots and sunken and circular pits (Hadidi and Barba, sample for DAS ELISA and RT-PCR. 2011). DAS ELISA. For the initial screening and detection of Plum plantations in Latvia include cultivars derived from ACLSV, PPV, ApMV, ArMV, PeAMV, PDV, PNRSV, L., such as traditionally grown cultivars SLRSV, ToRSV commercially available DAS ELISA kits ’Victoria’, ’Julius’, ‘Experimentalfältets Sviskon’, ’Pe- (Bioreba AG, Switzerland) were used according to the man- rdrigon’, ’Stanley’, and the more recently established dip- ufacturer’s instructions with some modifications. The coat- loid cultivars derived from crosses between P. salicina ing and conjugate conditions were changed to an overnight Lindl., P. cerasifera Ehrh., P. simonii Carrière and P. incubation in a refrigerator at 4–6 °C to increase sensitivity americana Marshall (e.g. cultivar ‘Kubanskaya Kometa’) of the test. The absorbance was read at 405/492 nm with a (Skrîvele et al., 2008; Kaufmane et al., 2010). Viral dis- dual filter microplate reader Asys Expert 96 (Hitech, Aus- eases in stone fruit orchards were not been studied until tria) after 30 min,1hand2hofincubation. Lyophilised 2008, when survey and screening on occurrence was initi- samples supplied in the kit for each virus were used as posi- ated in national research programmes (Pûpola et al., 2010). tive and negative controls. A “cut-off” value was calculated In other previous studies on stone fruit viruses in Latvia according to manufacturer recommendations (Bioreba AG, (Turka et al., 1984) only visual observations have been re- Switzerland). corded and noted as infections by ACLSV and ApMV. RNA extraction. For total RNA isolation, collected leaves Since a certification system for planting material has not were ground in liquid nitrogen until fine powder and ap- been established in Latvia, the risk for continuous spread of proximately 100 mg of each sample were suspended in 200 viral diseases in orchards is high. ml of TE buffer. The extraction of RNA was carried out Timely monitoring of virus infections and elimination of in- with the Genomic DNA Purification Kit (Thermo scientific, fected trees from orchards is a key issue in preventing the Lithuania) following the manufacturer’s recommendations spread of viruses. For routine diagnosis, reliable, fast and with minor modifications adapted for RNA isolation. Nu- inexpensive procedures are essential. Despite wide use in cleic acid precipitation was done with cold 96% ethanol routine and screening diagnosis, ELISA tests may fail due overnight at –20 °C instead of 10 min at –20 °C. After ex- to low viral titre and the inhibitory effect of woody plant traction DNase I (Thermo Scientific, Lithuania) was used to sap compounds (Kinard et al., 1996; MacKenzie et al., obtain DNA free RNA. The quantity and quality of the 1997). Therefore, PCR based techniques, which are more RNA was measured using a spectrophotometer NanoDropR sensitive and accurate, can provide an alternative for virus ND-1000 (Thermo Scientific, USA). RNA was directly detection in woody plants (Kinard et al., 1996; Sánchez- used for RT-PCR assays or stored at –80 °C until analysis. á Navarro et al., 2005; López et al., 2008; Jaroðov et al., RT-PCR. RT-PCR assay was applied for detection of Ç 2010; evik et al., 2011). ACLSV, ApMV, PDV, PNRSV and PPV. RT-PCR assays The aim of the research presented here was to determine the were carried out with a One-Step RT-PCR kit (Qiagen AG, occurrence of nine viruses of Prunus in Latvian plum or- Germany) in 50-µl reactions according to the manufac- chards, as well as to identify the PPV strains present and to turer’s instructions. All samples for ACLSV and the major- compare the detection rates by ELISA and RT-PCR for the ity of the samples for the other viruses were tested twice. most significant viruses. Primers used in this study are presented in Table 1. RT-PCR was carried out in a Mastercycler® thermocycler (Eppendorf AG, Germany). Reverse transcription was car- o MATERIALS AND METHODS ried out 30 min at 50 C, activation of the HotStart Taq DNA polymerase was at 95 oC for 15 min, followed by 40 Sampling and plant material. Samples of European plum cycles of 94 oC for 30 s, 55 oC for 45 s, 72 oC for 1 min, (P. domestica L.), cherry plum (P. cerasifera Ehrh.), Pru- and a final extension step at 72 oC for 10 min. Positive sam- nus inter-specific hybrids and other Prunus species were ples for each virus repeatedly detected by DAS ELISA were collected in May 2008. Ten fully expanded leaves were ran- selected and used as positive controls in RT-PCR. PCR domly collected around the canopy from each individual grade water was used as a quality control. PCR products tree and represented one sample. In total 654 leaf samples were separated by electrophoresis in 2% agarose gel in 1x (one sample/one tree) from 92 different genotypes, includ- TAE buffer, stained with ethidium bromide, and visualised ing different cultivars, hybrids and species, were collected under UV light. Approximate sizes of RT-PCR products

Proc. Latvian Acad. Sci., Section B, Vol. 67 (2013), No. 2. 117 Table 1 LIST OF THE PRIMERS USED IN THIS STUDY AND EXPECTED SIZES OF RT-PCR PRODUCTS FOR EACH PRIMER PAIR

Primer Sequence 5’to 3’ Amplicon size, bp Reference ApMVs CGTAGAGGAGGACAGCTTGG 450 Hassan et al., 2006 ApMVa CCGGTGGTAACTCACTCGTT ACLSVs TTCATGGAAAGACAGGGGCAA 677 Menzel et al., 2002 ACLSVa AAGTCTACAGGCTATTTATTATAAGTCTAA PDV-17F CGAAGTCTATTTCCGAGTGGATGC 303 Massart et al., 2008 PDV-12R CACTGGCTTGTTTCGCTGTGAAC PNRSV-10F TTCTTGAAGGACCAACCGAGAGG 348 Massart et al., 2008 PNRSV-10R GCTAACGCAGGTAAGATTTCCAAGC P1 ACCGAGACCACTACACTCCC 243 Wetzel et al., 1991b P2 CAGACTACAGCCTCGCCAGA PM* CTTCAACAACGCCTGTGCGT 198 Olmos et al., 1997 PD* CCTCAACGACACCCGTACGG

* Used as a reverse primer in pair with P1 primer. were determined with an O’RangeRuler 100 bp DNA ladder methods is shown in Figure 1. The other viruses tested by (Thermo scientific, Lithuania). DAS ELISA were detected only in a few cases. ArMV, ToRSV and SLRSV were detected in 0.6% of the trees and Data analyses. The data were analysed by Fisher’s exact PeAMV in 0.5% of the analysed samples. During the or- test using SPSS 15.0 computer software (SPSS Inc. USA). chard surveys no obvious symptoms were observed on plum The two-tailed Fishers’s exact test was used to determine trees, except in a few cases when characteristic chlorotic significance at P < 0.05 of the associations between vari- rings, necrotic spots and shot holes of PPV infection on ables. The occurrence of viruses was calculated as percent- plum leaves were observed (Fig. 2). The observed PPV age of positive samples out of the total tested samples. The symptoms were confirmed by RT-PCR and in all positive significance level of virus occurrence among cultivars was samples only the PPV-D strain was detected by use of s calculated by mean value (µ) ± standard deviation ( ). strain-specific primers.

The part of the data presented here on detection of ApMV, Positive analyses by RT-PCR resulted in almost a two times PDV, PNRSV by DAS ELISA and of ACLSV by RT-PCR higher number for PNRSV and PDV than those obtained by has been previously reported by Pûpola et al. (2010; 2011). DAS-ELISA (Fig. 1). In contrast, for ApMV, ACLSV and PPV, the detection rate with DAS ELISA was slightly RESULTS higher and the data obtained by both methods were in con- cordance for these viruses. In total, 38% of the tested sam- Detection of stone fruit viruses. Based on DAS ELISA ples by RT-PCR and 24% by DAS ELISA were detected and RT-PCR diagnosis, all of the studied viruses were de- positive for at least one of the tested viruses. Moreover, tected in plum orchards. The most widespread stone fruit vi- when DAS ELISA and RT-PCR results were directly com- ruses in plum trees were PNRSV and PDV. The occurrence pared one by one, not all samples that tested positive by of stone fruit viruses in plums tested by both diagnostic ELISA were detected positive by RT-PCR and vice versa.

Fig. 1. Occurrence (%) of stone fruit viruses in plums in Latvia assessed with one step RT-PCR and DAS ELISA. ACLSV, Apple chlorotic leaf spot virus; PDV, Prune dwarf virus; PNRSV, Prunus necrotic ringspot virus; ApMV, Apple mosaic virus; PPV, Plum pox virus.

118 Proc. Latvian Acad. Sci., Section B, Vol. 67 (2013), No. 2. Table 2 DIRECT COMPARISONS AND CONFORMITY OF POSITIVE AND NEGATIVE RESULTS OBTAINED WITH DAS ELISA AND RT-PCR

Virus Method Conformity ELISA* RT-PCR* %** positive negative positive negative positive negative ACLSV 17 637 13 641 76.5 98.9 PDV 71 583 107 547 66.4 93.8 PNRSV 83 571 201 453 41.3 79.3 ApMV 13 641 12 642 92.3 99.8 PPV 16 638 10 644 62.5 99.1

* Number of samples. ** The conformity of positive and negative results obtained by DAS ELISA and RT-PCR, calculated in percentages. ACLSV, Apple chlorotic leaf spot virus; PDV, Prune dwarf Virus; PNRSV, Prunus necrotic ringspot virus; ApMV, Apple mosaic virus; PPV, Plum pox virus.

Table 3 OCCURRENCE OF VIRUSES IN PLUMS IN THE GEOGRAPHICAL REGIONS OF LATVIA Fig. 2. Chlorotic rings and necrotic spots characteristic for Plum pox virus infection on plum hybrid 1256E. Region Total Number of positive samples, %** number of ApMV PDV PNRSV PPV ACLSV samples Direct comparison of positive and negative detection results Western part 52 2 40.4 30.8 0.0 2.0 obtained by DAS ELISA and RT-PCR are presented in Ta- Eastern part 149 4.7 43.6 51.0* 2.0 1.3 ble 2. Southern part 195 2.1 0.5 26.7 0.5 2.6* Northern part 69 0.0 2.9 7.2 4.3 1.4 Occurrence of mixed infections. Mixed infections were Central part 189 0.0 9.5 27.5 1.6 2.1 detected in 13.8% of the tested plum samples. The most common co-infection was PNRSV with PDV (10.9%). In * Indicates significant difference among the geographical regions by few cases ApMV was detected in combination with PDV Fishers exact test (P < 0.05) (0.5%), ApMV with PNRSV (1.2%) and ACLSV with ** Detected with RT-PCR PNRSV (1.2%). Mixed infections of three viruses were de- Abbreviations as in Table 2. tected only in few samples (0.2–0.3%). The detected combi- nations of three viruses were PNRSV+PDV+ACLSV, PDV+ApMV+PNRSV or PPV+ACLSV+PNRSV. Sin- found in all regions. ApMV was not found in the northern gle-virus infections were detected approximately in one- and central parts and was detected only in four orchards. third of the tested plum samples. Single infection with PPV was not detected in the western part and was found in PNRSV was predominant (17.4%) in comparison to other three out of 28 surveyed orchards; its occurrence in general infections and co-infections. Among the other tested vi- was low. ACLSV was found in six orchards and its occur- ruses, mainly only one virus was detected in the samples, rence was significantly higher in the southern part of the except in cultivar ‘Kubanskaya Kometa’ where infection country than in the rest. SLRSV was detected in samples with ArMV, ToRSV, SLRSV and PeAMV was detected. In from four orchards in the central and western parts, ToRSV cultivar ‘Skoroplodnaya’ ArMV, SLRSV and PeAMV were in four orchards in central and southern parts, ArMV in detected by DAS ELISA. three orchards in central, eastern and western parts, and PeAMV was detected in two orchards in the central and Occurrence of viruses in relation to orchards and geo- southern parts of the country. graphical regions. The most widespread virus, PNRSV, was detected in 16 out of 28 surveyed plum orchards. The Occurrence of viruses in relation to host genotypes. virus was detected in all regions, but its occurrence was sig- Among different plum genotypes a high variability in the nificantly higher in the eastern part of Latvia. Data on oc- occurrence of the viruses was observed (Table 4). PNRSV currence of stone fruit viruses in different geographical re- was detected in almost all commonly grown plum geno- gions in Latvia are shown in Table 3. A high PNRSV types, except in the case of Estonian origin cultivar ‘Julius’. occurrence was detected in all regions of Latvia, except in Cultivars ‘Aleynaya’, ‘Perdrigon’ and P. cerasifera Ehrh, the western part, where PDV was the predominant virus. were found significantly more infected with PNRSV than PDV was detected in 13 out of 28 surveyed orchards and the other plum genotypes. Similarly, cultivars ‘Aleynaya’,

Proc. Latvian Acad. Sci., Section B, Vol. 67 (2013), No. 2. 119 Table 4 ‘Skoroplodnaya’ (SLRSV, ToRSV and ArMV), ‘Washing- THE OCCURRENCE OF STONE FRUIT VIRUSES IN MOST COM- ton’ (ToRSV), ‘Reine Claude d’Oullins’ (ArMV) and in one MONLY GROWN PLUM GENOTYPES local cultivar ‘Lâse’ (PeAMV).

Genotype Total Number of infected samples (%)** number of PDV ApMV ACLSV PNRSV PPV samples DISCUSSION Aleynaja 12 41.7* 0 0 91.7* 0 The obtained results confirmed the presence of all tested vi- Ave 14 0 0 7.1 14.3 0 ruses in plum orchards in Latvia and showed that the major- Âþenas*** 11 9.1 0 18.2* 9.1 0 ity of commonly grown cultivars are infected either with Latvijas Dzeltenâ 31 21.2 3.0 0 36.4 0 PNRSV, PDV, ApMV, PPV and ACLSV or with their com- Olplûme binations at various degrees. ArMV, PeAMV, SLRSV and Prince of Wales 16 6.3 6.3 0 25.0 0 ToRSV were also detected in few cases by DAS ELISA. Experimentalfältets 24 20.8 0 16.7* 25.0 0 Although plums are known as a host of these viruses in Eu- Sviskon rope (Lovisolo, 1990; Diekmann and Putter, 1996; Jubileum 10 40.0* 0 0 40.0 0 Kommineni et al., 1998), they are not listed in EPPO stan- Juliu 19 5.3 0000 dards as requirements to be tested in certification schemes Kârsavas 9 0 33.3* 0 44.4 0 of plums. Although detection results of these viruses are in- Kubanskaya Kometa 64 20.3 1.6 1.6 23.4 0 dicative and need to be further verified with another test Lâse 28 0 3.6 3.6 28.6 3.58 method, e.g. RT-PCR, possibly they also need to be consid- ered in certification schemes of plums to prevent their fur- Mirabelle de Nancy 9 22.2 11.1 0 44.4 0 ther spread. Perdrigon 15 6.7 0 6.7 60.0* 13.3* Prunus cerasifera 9 55.6* 11.1 0 55.6* 0 In general, when compared to the occurrence of viral dis- Ehrh. eases in apple and pear in Latvia (Pûpola et al., 2011), the Skoroplodnaya 31 19.4 0 0 16.1 0 occurrence of viruses in plum orchards was relatively low, Stanley 18 5.6 0 0 16.7 0 as revealed in this study. The most widespread viruses de- Tragedy 11 0 0 0 18.2 0 tected in plum orchards were PNRSV and PDV. Both vi- Reine Claude 19 0 0 0 42.1 0 ruses are seed and pollen transmitted (Desvignes, 1999). In d’Oullins Latvia mainly seedlings of P. cerasifera Ehrh are used as Victoria 56 21.4 0 3.6 21.4 0 rootstocks for plum propagation (Kârkliòð et al., 2007). Our Mean 15.6 3.7 3.0 32.2 0.89 data on the occurrence of stone fruit viruses in different plum genotypes revealed that more than 50% of the tested * Indicates significantly higher occurrence over mean value among most P. cerasifera trees are infected with PNRSV and/or PDV. m s common plum genotypes ( + ). This indicates that most likely the main sources of inoculum ** Detected with RT-PCR. for the spread of PNRSV and PDV in plum cultivars have *** Cultivar of unknown origin is grown under this name in Latvia probably been infected rootstocks. (Kârkliòð et al., 2007). Abbreviations as in Table 2 Although in other countries PDV is reported as the most common Prunus virus (Gümüs`, 2007; Suchá, 2010), in our study on plums PNRSV was the most widespread. How- ever, according to the data from other studies, the occur- ‘Jubileum’ and P. cerasifera Ehrh were found significantly rence of PDV and PNRSV in different regions is highly more infected with PDV. PDV was less spread in plum ge- variable, ranging from 0.4% to 17% for PDV (Domínguez notypes than PNRSV and it was not detected in several lo- et al., 1998; Jarrar et al., 2001; Herrera and Madariaga, cal and introduced cultivars, such as ‘Ave’, ‘Kârsavas’, 2002) and 6% to 46% for PNRSV (Domínguez et al., 1998; ‘Lâse’, ‘Tragedy’ and ‘Reine Claude d’Oullins’. The occur- Jarrar et al. 2001; Herrera and Madariaga, 2002; Myrta et rence of ApMV, ACLSV and PPV was rather low in all al., 2002). common plum cultivars, except in the cases of cultivars ‘Kârsavas’, ‘Âþenas’ and ‘Experimentalfältets Sviskon’. The graft-transmitted viruses ACLSV and ApMV were not PPV was detected only in two cultivars among the most commonly detected, however, some of the plum cultivars common plum genotypes in the orchards and in the intro- showed more prevalence of these viruses than others. The duced cultivar ‘Perdrigon’ its occurrence was significantly local cultivar ‘Kârsavas’ frequently appeared to be infected higher. by ApMV. The Swedish origin cultivar ‘Experimentalfältets Sviskon’ and an unknown cultivar grown under the name of The other tested viruses by DAS ELISA were detected in a ‘Âþenas’, showed a high ACLSV prevalence, suggesting limited number of genotypes. They were detected in the in- that the original source of the material was probably already troduced cultivars ‘Prince of Wales’ (SLRSV), ‘Kuban- infected when introduced and that ApMV and ACLSV skaya Kometa’ (SLRSV, ToRSV, ArMV and PeAMV), spread by the use of this infected propagation material.

120 Proc. Latvian Acad. Sci., Section B, Vol. 67 (2013), No. 2. Although PPV has spread throughout several European fected by the low concentration of viruses in host plant and countries (Sochor et al., 2012), our study revealed that PPV the sampling time in the season (Torrance and Dolby, 1984; is not widely spread in Latvia and is currently present only Desvignes, 1999). in a limited number of genotypes and geographical locali- ties. One of the main reasons for the limited spread of PPV Our study demonstrated that stone fruit viruses are rela- in plum orchards in Latvia could be the low prevalence of tively not widespread in plum orchards in Latvia and that aphid species in Northern European countries (Verhoeven et the commonly grown genotypes are infected with one or al., 1998; Blystad and Munthe, 2006; Wijkamp and Gaag, more of the tested viruses. In the future, the implementation 2011). Most likely, the entry and establishment of the virus of a programme to produce and propagate virus free plant- occurred by infected bud-wood as suggested by the case of ing material and the establishment of virus free planting ma- the Western European origin cultivar ‘Perdrigon’, which is terial collections and certification programmes, which are one of the oldest introductions in Latvia (Kârkliòð et al., currently not established in the country, will play the key 2007). Moreover, in our study one of the most common role for the containment of the spread of these viruses in the PPV strains in Western Europe, the strain PPV-D, was de- orchards. tected in all of the PPV positive samples. The most recently discovered strain PPV-W (Winona), and also PPV-D, have ACKNOWLEDGEMENTS been reported in Latvia (Glasa et al., 2011; Malinowski et al., 2012). Our data confirm that actions should be taken The research was financed by the Latvia Ministry of Agri- and eradication continued to prevent further spread of this culture, National Subsidy project Nr. 030507/S92 (2008– detrimental , which currently is not widely 2009) and ESF project No. 2009/0228/1DP/1.1.1.2.0/09/ spread in the country. APIA/VIAA/035 (2010-2012).

Mixed infections of two or more viruses have frequently We are grateful to Latvian fruit growers for their coopera- been reported in stone fruit trees in different regions of the tion, to Mâris Jundzis for assistance during the surveys, to world (Mytra et al., 2003; Çevik et al., 2011). In our study Vitaliy Radenkov for help with statistical analyses and to mixed infections of two or three viruses were detected in our colleagues who helped with information. less than half of the positive samples. The combination of three viruses also occurred, but it was not common. The REFERENCES most commonly detected mixed infection was the combina- Anonymous (2004). PM 4/29 (1) Certification scheme for cherry. Bulletin tion of PNRSV with PDV as a logical consequence of their OEPP/ EPPO, 31, 447–462. transmission modes and of their wide distribution among Barba, M., Hadidi, A., Candresse, T., Cambra, M. (2011). Plum pox virus. In: the rootstock species sources and orchards. Hadidi, A., Barba, M., Candresse, T., Jelkmann, W. (eds.). Virus and Vi- rus-like Diseases of Pome and Stone Fruits (pp. 185–197). St. Paul, Minne- Comparison of DAS ELISA and RT-PCR as diagnostic sota: APS Press. methods for detection of economically important stone fruit Blystad, D. R., Munthe, T. (2006). Plum pox virus (PPV) in Norway. Bulletin viruses in plums resulted in considerable differences only OEPP/EPPO Bulletin, 36, 212. for PNRSV and PDV. For PNRSV and PDV, RT-PCR was Cambra, M., Capote, N., Myrta, A., Llácer, G. (2006). Plum pox virus and es- more effective and detected more positive samples. For timated costs associated with sharka disease. Bulletin OEPP/EPPO Bulle- ACLSV, ApMV and PPV results obtained by both methods tin, 36, 202–204. were in agreement with previously reported comparisons Capote, N., Bertolini, E., Olmos, A., Vidal, E., Martínez, M. C., Cambra, M. between ELISA and RT-PCR methods for PPV detection in (2009). Direct sample preparation methods for detection of Plum pox virus terms of specificity and sensitivity (Capote et al., 2009; by real-time RT-PCR. Int. Microbiol., 12, 1–6. Vidal et al. 2012a). Our results agree with data obtained in Çevik, B., Yardimci, N., Çulal-Klllç, H. (2011). Detection of viruses infect- other studies (Sánchez-Navarro et al., 1998; 2005; Çevik et ing stone fruits in Western Mediterranean region of Turkey. Plant Pathol. al., 2011) suggesting that RT-PCR seems to be more effec- J., 27 (1), 44–52. tive than ELISA, giving more positive reactions for the de- Desvignes, J. C. (1999). Virus Diseases of Fruit Trees. Paris: Centre tech- tection of PNRSV and PDV. However, in direct compari- niques interprofessionnel des fruits et legumes. 202 pp. sons one by one, not all positive ELISA samples were Diekmann, M., Putter, C. A. J. (1996). FAO/IPGRI technical guidelines for confirmed with RT-PCR and vice versa, probably due to the the safe movement of germplasm. In: Diekmann, M., Putter, C. A. J. (eds.). Stone Fruits, No. 16. (pp. 38–39). Rome: FAO Publication Division. presence of both false positives and negatives. Our study confirms that the use of only one diagnostic method is not Domínguez, S., Aparicio, F., Sánchez-Navarro, J., Cano, A., Pallás, V., í reliable and should be treated with caution due to the draw- Garc a-Brunton, J. (1998). Studies on the incidence of and Ap- ple chlorotic leaf spot virus (ACLSV) in apricot trees in the Murcia region backs of both methods, as discussed by Vidal et al., (2012a (Spain) using serological and molecular hybridization methods. Acta and b). As pointed out by MacKenzie et al. (1997) and Ca- Hort., 472, 203–210. pote et al. (2009), despite the potentially higher sensitivity García, J. A., Cambra, M. (2007). Plum pox virus and Sharka disease. Plant and specificity of PCR than ELISA, several factors, such as Viruses 1, 69–79. PCR inhibitors, RNA purity and primer specificity can in- Glasa, M., Candresse, T. (2005). Partial sequence analysis of an atypical fluence diagnostic performance and could give false-nega- Turkish isolate provides further information on the evolutionary history of tive results. Similarly, the sensitivity of ELISA can be af- Plum pox virus (PPV). Virus Res., 108, 199–206.

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KAULEÒKOKU VÎRUSU IZPLATÎBA PLÛMJU STÂDÎJUMOS LATVIJÂ Veikts pçtîjums, lai izvçrtçtu tâdu deviòu vîrusu, kuri ir sastopami Prunus ìints augiem, izplatîbu plûmju stâdîjumos Latvijâ. Âbeïu mozaîkas vîrusa (ApMV), plûmju pundurainîbas vîrusa (PDV), kauleòkoku nekrotiskâs gredzenplankumainîbas vîrusa (PNRSV), âbeïu hlorotiskâs lapu plankumainîbas vîrusa (ACLSV) un plûmju virâlo baku vîrusa (PPV) diagnostika veikta, izmantojot RT-PCR and DAS ELISA noteikðanas metodes, kâ arî veikts ðo abu metoþu salîdzinâjums. Zemeòu latentâ gredzenplankumainîbas vîrusa (SLRSV), Arabis mozaîkas vîrusa (ArMV), tomâtu gredzenplankumainîbas vîrusa (ToRSV) un petûniju asteroîdâs mozaîkas vîrusa (PeAMV) diagnostika veikta, izmantojot vienîgi DAS ELISA testu. Kopumâ 38% no visiem ar RT-PCR pârbaudîtajiem paraugiem bija inficçti vismaz ar vienu no testçtajiem vîrusiem. No tiem 30.7% bija inficçti ar PNRSV un 16.4 ar PDV, savukârt ApMV, ACLSV un PPV diagnosticçti nelielâ skaitâ paraugu. Visbieþâk sastopamâ vairâku vîrusu infekcija bija PNRSV kombinâcijâ ar PDV. Novçrotie raksturîgie PPV simptomi tika apstiprinâti laboratoriski ar RT-PCR un visos pozitîvajos paraugos noteikts PPV-D celms. Metoþu salîdzinâjums parâdîja, ka pielietoto RT-PCR un DAS ELISA metoþu efektivitâte vîrusu noteikðanai plûmju paraugos atkarîga no nosakâmâ vîrusa. Pçtîjuma rezultâtâ noteikts, ka plaðâk audzçtâs plûmju ðíirnes ir inficçtas ar ekonomiski nozîmîgiem kauleòkoku vîrusiem un norâda uz nepiecieðamîbu ieviest vîrusbrîva stâdâmâ materiâla audzçðanas sistçmu, lai novçrstu ðo vîrusu tâlâku izplatîðanos augïu dârzos.

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